G-protein-coupled receptors (GPCRs) represent a large protein family responsible for mediating extracellular to intracellular signaling. They are located in the plasma membrane of all cell types of various tissues and are involved in the control of numerous central and peripheral physiological responses, as well as being a major drug target in human disease. This family of receptors consists of muscarinic receptors designated as M1-M5. Each receptor is a single subunit membrane protein with seven α-helical transmembrane spanning domains, three extracellular and three intracellular loops which form a pocket for ligand binding. The gene family as a whole has approximately 26% overall amino acid identity. The third loop which selectively couples with G proteins is particularly variable, showing 2.7% identity between receptors compared with an average of 66% identity found in the conserved transmembrane domains. M2 and M4 receptors normally couple to the pertusiss-toxin sensitive Gi/o-proteins, whereas M1, M3 and M5 receptors couple to Gq/11-proteins. Smooth muscle contraction, particularly of airway, ileum, iris and bladder, is mediated primarily by M3 muscarinic receptors and to a much lesser extent by M2 receptors. Exocrine secretion, particularly of saliva and insulin, is primarily mediated by M3 receptors and to a smaller extent by M1 receptors. M2 receptors in the heart have a profound role in the control of cardiac myocyte contraction. M1 receptors have been found to play an important role in memory and learning while M4 receptors are thought to mediate an inhibitory effect on striatal dopamine-mediated locomotor activity. Additionally, M2 receptors located in the hypothalamus are likely to be involved in the regulation of body temperature whereas M3 receptors have been reported to modulate appetite. These muscarinic receptor subtypes thus provide effective therapeutic targets for a number of neurological and psychiatric diseases such as Alzheimer's (AD), Schizophrenia and Parkinson's Disease. Research has focused on the development of selective M1/M5 muscarinic agonists and M2 antagonists as potential therapeutic targets for the symptomatic treatment of AD, M1/M4 agonists for Schizophrenia, and M1/M4 antagonists for Parkinson's Disease. In addition to central nervous system (CNS) disorders, selective M2 and M3 muscarinic antagonists can be beneficial for the treatment of disorders such as chronic obstructive pulmonary disease (COPD), asthma and overactive bladder (OAB) syndrome.
Classical approaches to GPCR drug design have targeted the orthosteric receptor binding site and most drugs are known to interact with this endogenous ligand-binding pocket. Since the orthosteric site is usually conserved across GPCR subtypes, orthosteric ligands must overcome many limitations, such as decreased selectivity, efficacy, and undesirable side effects, thus making the development of subtype-specific agonists and antagonists very difficult. G-protein receptors also contain topographically distinct and less conserved allosteric binding sites that are targeted to attain drug selectivity. Novel drugs that modulate receptor function by targeting these allosteric sites alter the biological properties of the endogenous orthostheric ligand by either changing its affinity and/or efficacy. Allosteric compounds show higher subtype selectivity, have a safer pharmacological profile and often do not induce receptor desensitization. Allosteric modulators have therefore considerable therapeutic potential but their signaling properties are only beginning to be explored. Researchers have begun to explore the potential of linking orthosteric and allosteric pharmacophores to yield bitopic ligands. Bitopic ligands are single molecules with two different structural motifs chemically attached in a manner that allow simultaneous binding to both allosteric and orthosteric sites of the receptor. The goal is to combine high affinity, via orthosteric sites, with high selectivity, via allosteric sites. Bitopic ligands may lead to novel drugs with improved affinity and/or selectivity profiles, and muscarinic acetylcholine receptors (mAChRs).